The mononuclear phagocyte system is associated with a variety of functions, including host defense against microbial or neoplastic disease. Mononuclear phagocytes appear to be functionally heterogeneous. For example, some populations of mononuclear phagocytes are more easily activated for tumor cell killing than are others. There is little understanding of the role of differentiation in the origin of this diversity. The long range goal of our research is, therefore, to understand how differentiation and the functional heterogeneity of mononuclear phagocytes are related. To accomplish this goal, we will first determine how mononuclear phagocytes differentiate. The current application focuses on that objective. In particular, we will determine whether mononuclear phagocyte differentiation follows a single, linear pathway or, as with lymphocytes, a branched pathway. The research will proceed in four steps which coincide with the specific aims. Firstly, we will expand our present inventory of monoclonal antibodies produced against differentiating mouse mononuclear phagocytes. Secondly, antigens detected by monoclonal antibodies will be ranked in chronological order of their appearance using a combination of indirect fluorescence microscopy and flow cytometry. Thirdly, these antigens will be associated with specific stages of mononuclear phagocyte differentiation by three separate approaches: radioautography, cell sorting, and cytolytic depletion. Fourthly, we will determine if differentiation follows a linear or branched pathway. In the latter section our approach will be to determine, firstly, whether or not the expression of any antigen is clonally restricted. If clonal restriction is found, this will suggest the existence of a branching pathway of differentiation. Branches will then be localized along the differentiation pathway by a combination of cell sorting and standard cell cloning techniques. Results of the proposed studies will provide the foundations for future research aimed at determining whether specific functions, such as tumor cell killing, are associated with any of the subpopulations that we will have defined.